The Design of FluxML: A Universal Modeling Language for 13C Metabolic Flux Analysis

13C metabolic flux analysis (MFA) is the method of choice when a detailed inference of intracellular metabolic fluxes in living organisms under metabolic quasi-steady state conditions is desired. Being continuously developed since two decades, the technology made major contributions to the quantitative characterization of organisms in all fields of biotechnology and health-related research. 13C MFA, however, stands out from other “-omics sciences,” in that it requires not only experimental-analytical data, but also mathematical models and a computational toolset to infer the quantities of interest, i.e., the metabolic fluxes. At present, these models cannot be conveniently exchanged between different labs. Here, we present the implementation-independent model description language FluxML for specifying 13C MFA models. The core of FluxML captures the metabolic reaction network together with atom mappings, constraints on the model parameters, and the wealth of data configurations. In particular, we describe the governing design processes that shaped the FluxML language. We demonstrate the utility of FluxML to represent many contemporary experimental-analytical requirements in the field of 13C MFA. The major aim of FluxML is to offer a sound, open, and future-proof language to unambiguously express and conserve all the necessary information for model re-use, exchange, and comparison. Along with FluxML, several powerful computational tools are supplied for easy handling, but also to maintain a maximum of flexibility. Altogether, the FluxML collection is an “all-around carefree package” for 13C MFA modelers. We believe that FluxML improves scientific productivity as well as transparency and therewith contributes to the efficiency and reproducibility of computational modeling efforts in the field of 13C MFA

The study of RAS-induced metabolic reprogramming and the role of the pentose phosphate pathway in tumor metabolism

[spa] La presente tesis doctoral se centra en las adaptaciones metabólicas inducidas por la activación de oncogenes así como en el potencial del entramado metabólico como diana antitumoral. A lo largo de los últimos años, ha resurgido un renovado interés en el estudio del metabolismo, particularmente en el metabolismo de las células tumorales, dando lugar a una nueva disciplina conocida como metabolismo tumoral. Numerosas investigaciones se han centrado en la asociación entre mutaciones en oncogenes o genes supresores de tumores con perfiles metabólicos característicos, en busca de dependencias metabólicas que ofrezcan nuevas posibilidades para el tratamiento de los tumores. La búsqueda de alteraciones metabólicas que constituyan vulnerabilidades de la célula tumoral representa la piedra angular de esta interesante disciplina. Así, esta tesis doctoral tiene como objetivo general elucidar las alteraciones metabólicas que acompañan a la mutación de oncogenes y explorar el potencial del entramado metabólico como diana antitumoral. Por tanto, los objetivos principales de este trabajo son los siguientes: i) análisis de la reprogramación metabólica inducida por la activación oncogénica de RAS empleando líneas celulares transfectadas de manera estable con copias mutadas de los oncogenes K-RAS y H-RAS y, ii) validación de la vía de las pentosas fosfato como potencial diana antitumoral y estudio de su papel en el metabolismo tumoral de modelos celulares de cáncer de colon y de mama. Así, en este trabajo de tesis doctoral hemos concluido que la activación oncogénica de RAS promueve una profunda reprogramación del metabolismo induciendo cambios significativos en la glucólisis, la vía de las pentosas fosfato, el metabolismo de la glutamina y la lipogénesis. Por otro lado, hemos determinado que la inhibición de la vía de las pentosas fosfato tiene distintos efectos según el tipo de tumor. La inhibición de la G6PD en la línea celular de cáncer colon HT29 no produjo efectos sobre la proliferación mientras que su inhibición en células de cáncer de mama MCF7 indujo una notable reducción de la proliferación y un incremento de la muerte celular. Por otra parte, en la inhibición en MCF7 del otro enzima clave de la vía de las pentosas fosfato, la TKT, no se observaron cambios significativos en términos de proliferación y viabilidad celular. Además, en este trabajo también se ha puesto de manifiesto una conexión funcional entre la vía de las pentosas fosfato y el metabolismo de la glutamina en ambos modelos celulares, sugiriendo un papel complementario de estas dos vías metabólicas.[eng] The present doctoral thesis is focused on the metabolic adaptations induced by oncogene activation as well as the potential role of the metabolic network as antitumor therapy. Over the last years, it has emerged a renewed interest in the field of metabolism, particularly in cancer metabolism. Great efforts have been focused on the association of mutated oncogenes or tumor suppressor genes and tumor metabolic profiles, in the search of metabolic dependencies that offer new potential avenues for cancer treatment. The pursuit of discovering tumor metabolic alterations in which cancer cells rely on has represented the cornerstone of this interesting discipline. Thus, this thesis is part of this recent and promising scientific current and is intended to shed light on the metabolic alterations accompanying oncogene mutation and on potential metabolic pathways that might be of therapeutic interest in the future. Hence, the objectives of this thesis can be divided into two specific aims: i) analysis of the metabolic reprogramming of RAS oncogenic activation using stable transfected cell lines with mutated copies of K-RAS and H-RAS and ii) validation of the pentose phosphate pathway as a potential therapeutic target and exploration of its role within tumor metabolism in colon and breast cancer cell models. Thus, according to the proposed objectives, the main conclusions obtained are as follow: 1. The study of flux distribution in combination with metabolic control analysis performed by analyzing solely the sign of fixed-sign control coefficients, is a reliable approach to identify the key enzymes involved in metabolic reprogramming. The use of this methodology has allowed us to identify an increase in glycolysis and PPP fluxes as metabolic features of KRAS-induced metabolic reprogramming and to propose G6PD, PK and LDH as the key enzymes responsible for this metabolic transition. 2. H-RAS oncogenic activation reprograms glucose and glutamine metabolism by enhancing glycolytic and PPP fluxes as well as mitochondrial metabolism. Glutamine is responsible for sustaining the activated mitochondrial metabolism in BJ-HRasV12, while glucose-derived carbons in the mitochondria are primarily used to fuel lipogenesis. Moreover, lipogenesis is overactivated in BJ-HRasV12 cells, which are more sensitive to FAS inhibition than BJ cells. 3. G6PD enzyme is overactivated in colon cancer cells with oncogenic activation of the RAS signaling pathway. Nevertheless, G6PD seems to be dispensable for proliferation and survival in BRAF-mutated HT29 cell line. Furthermore, a new connection between PPP and glutamine metabolism has been unveiled, as G6PD is overexpressed in HT29 cells under glutamine-deprived conditions by a mechanism involving a concomitantly increase in ROS levels and NRF2 induction. 4. G6PD enzyme is important in proliferation, survival and regulation of ROS levels in breast cancer MCF7 cells. However, it exerts a low regulation over ribose synthesis flux through the oxidative branch of PPP. G6PD inhibition enhances glycolytic flux, promotes lactate secretion and increases glutamine consumption, which is used to maintain energy homeostasis, although it is not essential for cell proliferation. 5. TKT enzyme is dispensable for proliferation of breast cancer MCF7 cells, but it exerts a high control over ribose synthesis flux through the nonoxidative branch of PPP. TKT impairment reduces glycolytic flux and increases the consumption of glutamine, which is intended to maintain energy homeostasis but it is not essential for cell proliferatio

Tracer and Timescale Methods for Passive and Reactive Transport in Fluid Flows

Geophysical, environmental, and urban fluid flows (i.e., flows developing in oceans, seas, estuaries, rivers, aquifers, reservoirs, etc.) exhibit a wide range of reactive and transport processes. Therefore, identifying key phenomena, understanding their relative importance, and establishing causal relationships between them is no trivial task. Analysis of primitive variables (e.g., velocity components, pressure, temperature, concentration) is not always conducive to the most fruitful interpretations. Examining auxiliary variables introduced for diagnostic purposes is an option worth considering. In this respect, tracer and timescale methods are proving to be very effective. Such methods can help address questions such as, "where does a fluid-born dissolved or particulate substance come from and where will it go?" or, "how fast are the transport and reaction phenomena controlling the appearance and disappearance such substances?" These issues have been dealt with since the 19th century, essentially by means of ad hoc approaches. However, over the past three decades, methods resting on solid theoretical foundations have been developed, which permit the evaluation of tracer concentrations and diagnostic timescales (age, residence/exposure time, etc.) across space and time and using numerical models and field data. This book comprises research and review articles, introducing state-of-the-art diagnostic theories and their applications to domains ranging from shallow human-made reservoirs to lakes, river networks, marine domains, and subsurface flow

Microbial aspects of anaerobic methane oxidation with sulfate as electron acceptor

Anaerobic oxidation of methane (AOM) is an important methane sink in the ocean but the microbes responsible for AOM are as yet resilient to cultivation. It was shown that AOM was coupled to sulfate reduction (SR) and this gave rise to current research which aims to develop a biotechnological process in which methane is used an electron donor for SR. This thesis describes the microbial analysis of an enrichment capable of high rate AOM (286 µmol.gdry weight-1.day-1) coupled to SR using a novel submerged membrane bioreactor system. Initially AOM rates were extremely low (0.004 mmol L-1 d-1), but AOM and SR increased exponential over the course of 884 days to 0.60 mmol L-1 d-1. The responsible organisms doubled every 3.8 months. By constructing a clone library with subsequent sequencing and fluorescent in situ hybridization (FISH), we showed that the responsible methanotrophs belong to the ANME-2a subgroup of anaerobic methanotrophic archaea, and that sulfate reduction is most likely performed by sulfate reducing bacteria commonly found in association with other ANME related archaea in marine sediments. Another relevant portion of the bacterial sequences can be clustered within the order of Flavobacteriales but their role remains to be elucidated. FISH analyses showed that the ANME-2a cells occur as single cells without close contact to the bacterial syntrophic partner. Incubation with 13C labeled methane showed substantial incorporation of 13C label in the bacterial C16 fatty acids (bacterial; 20, 44 and 49%) and in archaeal lipids, archaeol and hydroxyl-archaeol (21 and 20%, respectively). This confirms that both archaea and bacteria are responsible for the anaerobic methane oxidation in a bioreactor enrichment inoculated with Eckernförde bay sediment. To unravel the pathway of this syntrophic conversion, the effect of possible intermediates on AOM and SR was assessed. To investigate which kind of waste and process streams can be treated by the methanotrophic sulfate-reducing enrichment, the effect of environmental conditions and different substrates was assessed. The optimum pH, salinity and temperature for SR with methane by the enrichment were 7.5, 30‰ and 20°C, respectively. The biomass had a good affinity for sulfate (Km 75 KPa) and AOM was completely inhibited at 2.4 (±0.1) mM sulfide. The enrichment utilized sulfate, thiosulfate, sulfite and elemental sulfur as alternative electron acceptors for methane oxidation and formate, acetate and hydrogen as alternative electron donors for sulfate reduction. As a co-substrate for methane oxidation only methanol stimulated the conversion of 13C labeled CH4 to 13CO2 in batch incubations of Eckernförde bay sediment, other possible co-substrates had a negative effect on the AOM rate. The research described in this thesis shows the possibility of enriching slow growing methane oxidizing communities but also shows the difficulties in applying this process for a biotechnological purpose because of the extreme slow doubling times and the lack of understanding of the metabolic routes used by these organisms. <br/

Molecular Imaging

The present book gives an exceptional overview of molecular imaging. Practical approach represents the red thread through the whole book, covering at the same time detailed background information that goes very deep into molecular as well as cellular level. Ideas how molecular imaging will develop in the near future present a special delicacy. This should be of special interest as the contributors are members of leading research groups from all over the world

Abstracts of manuscripts submitted in 1993 for publication

This volume contains the abstracts of manuscripts submitted for publication during calendar year 1993 by the staff and students of the Woods Hole Oceanographic Institution. We identify the journal of those manuscripts which are in press or have been published. The volume is intended to be informative, but not a bibliography. The abstracts are listed by title in the Table of Contents and ar grouped into one of our five departents, Marine Policy Center, Coastal Research Center, or the student category. An author index is presented in the back to facilitate locating specific papers

Methane Emissions From Lakes In Northeast Siberia And Alaska

Thesis (Ph.D.) University of Alaska Fairbanks, 2006Large uncertainties in the budget of atmospheric methane (CH4), an important greenhouse gas whose relative greenhouse effect is 23 times stronger than that of carbon dioxide (CO2), limit the accuracy of climate-change projections. Concentrations of atmospheric CH 4 have been rising during recent decades, particularly at high northern latitudes. The causes of this increase are not well understood. Here I describe and quantify an important source of methane---bubbling from northern lakes---that has not been incorporated in previous regional or global methane budgets. I introduce a new method to accurately measure ebullition (bubbling), which accounted for 95% of CH4 emissions from North Siberian thaw lakes. Documenting the patchiness of ebullition increased previous estimates of CH4 flux from lakes 5-fold in Siberia and 2.5- to 14-fold in Alaska. Extrapolating estimates of measured fluxes, I show that North Siberian yedoma (Pleistocene-aged organic-rich loess) thaw lakes emit 3.8 Tg CH 4 yr-1. An independent mass-balance approach based on carbon lost from permafrost that thawed beneath lakes revealed that lakes emit 4-5 Tg CH4 yr-1. Adding these emissions significantly increases present estimates of northern wetland contributions (<6-40 Tg yr-1) to the atmospheric CH4 budget. Thermokarst (thaw) erosion was the primary driver of CH4 emissions in lakes. A 14.7% expansion of thaw lakes from 1974 to 2000 increased lake CH 4 emissions by 58% in Siberia, demonstrating a positive feedback to climate warming. The Pleistocene age of CH4 (14C age 35,570-42,800 years in Siberia and 14,760-26,020 years in Alaska) emitted from hotspots along active thermokarst margins of lakes demonstrated that recruitment of a previously sequestered carbon source contributes to this feedback. Finally, reconstruction of yedoma's distribution at the Last Glacial Maximum together with compilation of thaw lake basal ages that developed at the onset of Holocene warming, suggested that thaw lake development contributed up to 70% of the rapid increase in atmospheric CH4 during deglaciation. About 425 Gt C remain preserved in the yedoma ice complex in North Siberia. If this Siberian permafrost warms more rapidly in the future as projected, the positive feedback of ebullition from expanding thaw lakes could increase the rate of high-latitude warming

Environmental Sciences Division: Summaries of research in FY 1996

This document describes the Fiscal Year 1996 activities and products of the Environmental Sciences Division, Office of Biological and Environmental Research, Office of Energy Research. The report is organized into four main sections. The introduction identifies the basic program structure, describes the programs of the Environmental Sciences Division, and provides the level of effort for each program area. The research areas and project descriptions section gives program contact information, and provides descriptions of individual research projects including: three-year funding history, research objective and approach used in each project, and results to date. Appendixes provide postal and e-mail addresses for principal investigators and define acronyms used in the text. The indexes provide indexes of principal investigators, research institutions, and keywords for easy reference. Research projects are related to climatic change and remedial action